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[Breath methane in children with chronic constipation].

RATIONALE: Methane is an intestinal gas which may be excreted in the expired air of about 10% of children. OBJECTIVE: The aims of this study were to investigate methane production by children with functional chronic constipation and methane concentration in the expired air before and after a bowel movement induced by a phosphate enema. METHODS: Seventy-five patients with functional chronic constipation aged from 3 to 13 years were studied. Methane concentration in the expired air was determined using a gas chromatograph (Quintron, model 12i). Methane production was considered present if the breath methane concentration was equal or greater than 3 ppm. RESULTS: Methane production was present in 44 (86.3%) of 51 patients with constipation and fecal soiling versus only 7 (29.2%) of 24 patients with constipation without fecal soiling. After six weeks of therapy for constipation, the number of methane producers decreased by 65.2%. None of the 10 children with normal intestinal habit produced methane. Expired air methane concentration was determined before and after a bowel movement induced by a phosphate enema in 20 patients with impacted stool. From these 20 patients, 12 were methane producers. The median (percentiles 25 and 75 between parenthesis) of methane concentration decreased from 21.5 (15.0-25.5) ppm before to 11.0 (4.0-12.5) ppm after the bowel movement. CONCLUSION: Methane production was associated with chronic constipation with soiling and decreased when impacted stool decreased.

Adolescent↗

Anaerobic methane oxidation: occurrence and ecology.

Anoxic sediments and digested sewage sludge anaerobically oxidized methane to carbon dioxide while producing methane. This strictly anaerobic process showed a temperature optimum between 25 and 37 degrees C, indicating an active microbial participation in this reaction. Methane oxidation in these anaerobic habitats was inhibited by oxygen. The rate of the oxidation followed the rate of methane production. The observed anoxic methane oxidation in Lake Mendota and digested sewage sludge was more sensitive to 2-bromoethanesulfonic acid than the simultaneous methane formation. Sulfate diminished methane formation as well as methane oxidation. However, in the presence of iron and sulfate the ratio of methane oxidized to methane formed increased markedly. Manganese dioxide and higher partial pressures of methane also stimulated the oxidation. The rate of methane oxidation in untreated samples was approximately 2% of the CH(4) production rate in Lake Mendota sediments and 8% of that in digested sludge. This percentage could be increased up to 90% in sludge in the presence of 10 mM ferrous sulfate and at a partial pressure of methane of 20 atm (2,027 kPa).

Journal Article↗

Mechanistic analysis of ammonium inhibition of atmospheric methane consumption in forest soils.

Methane consumption by forest soil was studied in situ and in vitro with respect to responses to nitrogen additions at atmospheric and elevated methane concentrations. Methane concentrations in intact soil decreased continuously from atmospheric levels at the surface to 0.5 ppm at a depth of 14 cm. The consumption rate of atmospheric methane in soils, however, was highest in the 4- to 8-cm depth interval (2.9 nmol per g of dry soil per day), with much lower activities below and above this zone. In contrast, extractable ammonium and nitrate concentrations were highest in the surface layer (0 to 2 cm; 22 and 1.6 mumol per g of dry soil, respectively), as was potential ammonium-oxidizing activity (19 nmol per g of dry soil per day). The difference in zonation between ammonium oxidation and methane consumption suggested that ammonia-oxidizing bacteria did not contribute significantly to atmospheric methane consumption. Exogenous ammonium inhibited methane consumption in situ and in vitro, but the pattern of inhibition did not conform to expectations based on simple competition between ammonia and methane for methane monooxygenase. The extent of ammonium inhibition increased with increasing methane concentration. Inhibition by a single ammonium addition remained constant over a period of 39 days. In addition, nitrite, the end product of methanotrophic ammonia oxidation, was a more effective inhibitor of methane consumption than ammonium. Factors that stimulated ammonium oxidation in soil, e.g., elevated methane concentrations and the availability of cosubstrates such as formate, methanol, or beta-hydroxybutyrate, enhanced ammonium inhibition of methane oxidation, probably as a result of enhanced nitrite production.

Journal Article↗

Evaluation of methane emissions from Taiwanese paddies.

The main greenhouse gases are carbon dioxide, methane and nitrous oxide. Methane is the most important because the warming effect of methane is 21 times greater than that of carbon dioxide. Methane emitted from rice paddy fields is a major source of atmospheric methane. In this work, a methane emission model (MEM), which integrates climate change, plant growth and degradation of soil organic matter, was applied to estimate the emission of methane from rice paddy fields in Taiwan. The estimated results indicate that much methane is emitted during the effective tillering and booting stages in the first crop season and during the transplanting stage in the second crop season in a year. Sensitivity analysis reveals that the temperature is the most important parameter that governs the methane emission rate. The order of the strengths of the effects of the other parameters is soil pH, soil water depth (SWD) and soil organic matter content. The masses of methane emitted from rice paddy fields of Taiwan in the first and second crop seasons are 28,507 and 350,231 tons, respectively. The amount of methane emitted during the second crop season is 12.5 times higher than that emitted in the first crop season. With a 12% reduction in planted area during the second crop season, methane emission could be reduced by 21%. In addition, removal of rice straw left from the first crop season and increasing the depth of flooding to 25 cm are also strategies that could help reduce annual emission by up to 18%.

Agriculture↗

Methane production and bowel function parameters in healthy subjects on low- and high-fiber diets.

Colon cancer patients are more likely than healthy subjects to excrete breath methane, and methane production has been suggested as a screening method to predict risk of developing colon cancer. To assess whether methane production is related to fiber ingestion and bowel function, we measured end-alveolar breath methane in 126 healthy subjects; 36% produced methane (greater than 3 ppm). Fifteen methane producers and nine controls were selected to participate in a 17-day study: a self-selected diet for 7 days, followed by the same diet plus 24 g/day dietary fiber (Fiber One Cereal) for 10 days. Methane was measured three times per day on three days during each diet. Two subjects screened as methane producers stopped excreting methane during the study, and one control started to excrete methane. Radiopaque pellets were given at the start and end of each diet, and feces were collected, homogenized, and dried. Mean transit time (p less than 0.0001) and fecal pH (p less than 0.0003) were significantly decreased with fiber, whereas fecal hydration scale (p less than 0.003), fecal dry (p less than 0.0001) and wet (p less than 0.0001) weight, and frequency of defecation (p less than 0.0006) were significantly increased with fiber. No significant change in methane excretion (p less than 0.057) was seen with added fiber. Thus, in this study methane excretor status was not constant in healthy subjects and was not significantly changed by 24 g/day dietary fiber as Fiber One Cereal.

Adolescent↗

Methane emissions from cattle.

Increasing atmospheric concentrations of methane have led scientists to examine its sources of origin. Ruminant livestock can produce 250 to 500 L of methane per day. This level of production results in estimates of the contribution by cattle to global warming that may occur in the next 50 to 100 yr to be a little less than 2%. Many factors influence methane emissions from cattle and include the following: level of feed intake, type of carbohydrate in the diet, feed processing, addition of lipids or ionophores to the diet, and alterations in the ruminal microflora. Manipulation of these factors can reduce methane emissions from cattle. Many techniques exist to quantify methane emissions from individual or groups of animals. Enclosure techniques are precise but require trained animals and may limit animal movement. Isotopic and nonisotopic tracer techniques may also be used effectively. Prediction equations based on fermentation balance or feed characteristics have been used to estimate methane production. These equations are useful, but the assumptions and conditions that must be met for each equation limit their ability to accurately predict methane production. Methane production from groups of animals can be measured by mass balance, micrometeorological, or tracer methods. These techniques can measure methane emissions from animals in either indoor or outdoor enclosures. Use of these techniques and knowledge of the factors that impact methane production can result in the development of mitigation strategies to reduce methane losses by cattle. Implementation of these strategies should result in enhanced animal productivity and decreased contributions by cattle to the atmospheric methane budget.

Animal Feed↗

[The processes of methane formation and oxidation in the soils of the Russian arctic tundra].

Methane emission from the following types of tundra soils was studied: coarse humic gleyey loamy cryo soil, peaty gley soil, and peaty gleyey midloamy cryo soil of the arctic tundra. All the soils studied were found to be potential sources of atmospheric methane. The highest values of methane emission were recorded in August at a soil temperature of 8-10 degrees C. Flooded parcels were the sources of atmospheric methane throughout the observation period. The rates of methane production and oxidation in tundra soils of various types at 5 and 15 degrees C were studied by the radioisotope method. Methane oxidation was found to occur in bog water, in the green part of peat moss, and in all the soil horizons studied. Methane formation was recorded in the horizons of peat, in clay with plant roots, and in peaty moss dust of the bogey parcels. At both temperatures, the methane oxidation rate exceeded the rate of methane formation in all the horizons of the mossy-lichen tundra and of the bumpy sinkhole complex. Methanogenesis prevailed only in a sedge-peat moss bog at 15 degrees C. Enrichment bacterial cultures oxidizing methane at 5 and 15 degrees C were obtained. Different types of methanotrophic bacteria were shown to be responsible for methane oxidation under these conditions. A representative of type I methylotrophs oxidized methane at 5 degrees C, and Methylocella tundrae, a psychroactive representative of an acidophilic methanotrophic genus Methylocella, at 15 degrees C.

Arctic Regions↗

Molecular genetics of methane oxidation.

Biological methane oxidation is carried out by methanotrophs, bacteria that utilize methane as their sole carbon and energy source. The enzyme they contain that is responsible for methane oxidation is methane monooxygenase, the most well studied being the soluble methane monooxygenase enzyme complexes from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. In both organisms, the genes encoding soluble methane monooxygenase have been found to be clustered on the chromosome in the order mmoX, mmoY, mmoB, mmoZ, orfY and mmoC. These genes encode the alpha and beta subunits of Protein A, Protein B, the gamma subunit of Protein A, a protein of unknown function and Protein C respectively of the soluble methane monooxygenase complex. The complete DNA sequences of both gene clusters have been determined and they show considerable homology. Expression of soluble methane monooxygenase genes occurs under growth conditions where the copper-to-biomass ratio is low. Transcriptional regulation of the gene cluster from Methylosinus occurred at an RpoN-like promoter, 5' of the mmoX gene. mmoB and mmoC of Methylococcus have been expressed in E. coli and the proteins obtained were functionally active. Soluble methane monooxygenase mutants have been constructed by marker-exchange mutagenesis. They were found to be more stable than those generated using the suicide substrate dichloromethane. Soluble methane monooxygenase probes have been used to detect both methane monooxygenase gene-specific DNA and methanotrophs in natural environmental samples.

Base Sequence↗

Effect of natural zeolite on methane production for anaerobic digestion of ammonium rich organic sludge.

The effect of an inorganic additive on the methane production from NH(4+)-rich organic sludge during anaerobic digestion was investigated using different kinds of inorganic adsorbent zeolites (mordenite, clinoptilolite, zeolite 3A, zeolite 4A), clay mineral (vermiculite), and manganese oxides (hollandite, birnessite). The additions of inorganic materials resulted in significant NH4+ removals from the natural organic sludge ([NH4+]=1, 150 mg N/l), except for the H-type zeolite 3A and birnessite. However, an enhanced methane production was only achieved using natural mordenite. Natural mordenite also enhanced the methane production from the sludge with a markedly high NH4+ concentration (4500 mg N/l) during anaerobic digestion. Chemical analyses of the sludge after the digestion showed considerable increases in the Ca2+ and Mg2+ concentrations in the presence of natural mordenite, but not with synthetic zeolite 3A. The effect of Ca2+ or Mg2+ addition on the methane production was studied using Na(+)-exchanges mordenite and Ca2+ or Mg(2+)-enriched sludge. The simultaneous addition of Ca2+ ions and Na(+)-exchanged mordenite enhanced the methane production; the amount of produced methane was about three times greater than that using only the Na(+)-exchanged mordenite. In addition, comparing the methane production by the addition of natural mordenite or Ca2+ ions, the methane production with natural mordenite was about 1.7 times higher than that with only Ca2+ ions. The addition of 5% and 10% natural mordenite were suitable condition for obtaining a high methane production. These results indicated that the Ca2+ ions, which are released from natural mordenite by a Ca2+/NH4+ exchange, enhanced the methane production of the organic waste at a high NH4+ concentration. Natural mordenite has a synergistic effect on the Ca2+ supply as well on the NH4+ removal during anaerobic digestion, which is effective for the mitigation of NH4+ inhibition against methane production.

Bacteria, Anaerobic↗

Performance of a passively vented field-scale biofilter for the microbial oxidation of landfill methane.

An upflow biofilter system was operated on a passively vented landfill for the treatment of residual landfill methane. Biofilter methane emissions as a basis for determining methane removal rates were assessed by manual and automated chamber measurements, by measuring methane concentrations in the top layer gaseous phase in combination with gas flow rates, and by evaluating the methane load in the reverse gas flow following the change of landfill gas flux direction as governed by the course of barometric pressure. Methane removal rates were very high with maximum values of 80 g h(-1) m(-3). For the observed cases, the limit of biofilter methane oxidation capacity was not reached and absolute removal rates were thus linearly correlated to the amount of methane entering the filter. The analysis of methane loads flowing back from the biofilter following phases of longer, continuous and non-oscillating landfill gas emission, however, revealed that in these situations biofilter performance is restricted by deficient oxygen supply. At the oxygen-restricted capacity limit, removal rates are influenced by temperature (positively), methane influx (negatively) and flow rate (negatively) as a measure for the displacement of oxygen. These situations, however, account for only 12% of all emission phases. The investigated biofilter capacity, as derived from laboratory analyses of methanotrophic activities, is sufficient to oxidise 62% of the methane load emitted annually. Field and laboratory data provide a stable basis for the dimensioning of filters in future applications.

Air Pollutants↗

Methane bubbling from Siberian thaw lakes as a positive feedback to climate warming.

Large uncertainties in the budget of atmospheric methane, an important greenhouse gas, limit the accuracy of climate change projections. Thaw lakes in North Siberia are known to emit methane, but the magnitude of these emissions remains uncertain because most methane is released through ebullition (bubbling), which is spatially and temporally variable. Here we report a new method of measuring ebullition and use it to quantify methane emissions from two thaw lakes in North Siberia. We show that ebullition accounts for 95 per cent of methane emissions from these lakes, and that methane flux from thaw lakes in our study region may be five times higher than previously estimated. Extrapolation of these fluxes indicates that thaw lakes in North Siberia emit 3.8 teragrams of methane per year, which increases present estimates of methane emissions from northern wetlands (< 6-40 teragrams per year; refs 1, 2, 4-6) by between 10 and 63 per cent. We find that thawing permafrost along lake margins accounts for most of the methane released from the lakes, and estimate that an expansion of thaw lakes between 1974 and 2000, which was concurrent with regional warming, increased methane emissions in our study region by 58 per cent. Furthermore, the Pleistocene age (35,260-42,900 years) of methane emitted from hotspots along thawing lake margins indicates that this positive feedback to climate warming has led to the release of old carbon stocks previously stored in permafrost.

Atmosphere↗

Breath methane production in children with constipation and encopresis.

Breath methane excretion is uncommon in children compared with adults. Certain intracolonic conditions, however, have been associated with enhanced methane generation. We hypothesized that encopretic and constipated children, who have abnormal colonic transit times, more likely would excrete methane than healthy children. To determine the prevalence of methane excretion among children with encopresis or simple constipation, we performed breath methane analysis on such patients and age-, race-, and sex-matched control subjects. Encopretic patients (mean age, 8.3 +/- 3.0 years) had daily, involuntary passage of feces and clinical evidence of constipation. Constipated patients (mean age, 7.1 +/- 2.9 years) had a history of hard stools and at least one of the following symptoms: infrequent defecation, dyschezia, hematochezia, difficult stool expulsion, or abdominal pain during bowel movements. Methane excretion was present in 26 of 40 (65%) encopretic patients versus 6 of 40 (15%) control patients (P less than 0.001). In contrast, 3 of 27 (11%) constipated patients were methane excreters, versus 2 of 27 (7%) controls (P = 0.4). Fourteen asymptomatic encopretic patients were retested after successful therapy; eight were methane excreters initially, but five of eight did not excrete methane after treatment. We conclude that methane is produced in a large number of children with encopresis. Treatment appears to alter methanogenesis in such patients. The prevalence of methane producers among constipated children is not different from the prevalence in healthy subjects. Methanogenesis in encopretic patients may be enhanced by prolonged colonic transit time or abnormal intracolonic conditions.

Age Factors↗

Oxidation of methane in the absence of oxygen in lake water samples.

Methane was oxidized to carbon dioxide in the absence of oxygen by water samples from Lake Mendota, Madison, Wis. The anaerobic oxidation of methane did not result in the assimilation of carbon from methane into material precipitable by cold 10% trichloracetic acid. Only samples taken at the suface of the sediment of Lake Mendota were capable of catalyzine the anaerobic oxidation of methane. The rate of methane oxidation in the presence of oxygen was highest in samples taken from near the thermocline. Of the radioactive methane oxidized, 30 to 60% was assimilated into material precipitable by cold 10% trichloroacetic acid during aerobic incubation of the samples. These data support the conclusion that two distinct groups of methane-oxidizing organisms occur in stratifield lakes. Enrichments with acetate and methane as the sole sources of carbon and energy and sulfate as the electron acceptor resulted in the growth of bacteria that oxidize methane. Sulfate, acetate, and methane were all required for growth of enrichments. Acetate was not oxidized to carbon dioxide but was assimilated by cells. Methane was not assimilated but was oxidized to carbon dioxide in the absence of air.

Acetates↗

The regulation of methane oxidation in soil.

The atmospheric concentration of methane, a greenhouse gas, has more than doubled during the past 200 years. Consequently, identifying the factors influencing the flux of methane into the atmosphere is becoming increasingly important. Methanotrophs, microaerophilic organisms widespread in aerobic soils and sediments, oxidize methane to derive energy and carbon for biomass. In so doing, they play an important role in mitigating the flux of methane into the atmosphere. Several physico-chemical factors influence rates of methane oxidation in soil, including soil diffusivity; water potential; and levels of oxygen, methane, ammonium, nitrate, nitrite, and copper. Most of these factors exert their influence through interactions with methane monooxygenase (MMO), the enzyme that catalyzes the reaction converting methane to methanol, the first step in methane oxidation. Although biological factors such as competition and predation undoubtedly play a role in regulating the methanotroph population in soils, and thereby limit the amount of methane consumed by methanotrophs, the significance of these factors is unknown. Obtaining a better understanding of the ecology of methanotrophs will help elucidate the mechanisms that regulate soil methane oxidation.

Bacteria↗

Attenuation of methane and volatile organic compounds in landfill soil covers.

The potential for natural attenuation of volatile organic compounds (VOCs) in landfill covers was investigated in soil microcosms incubated with methane and air, simulating the gas composition in landfill soil covers. Soil was sampled at Skellingsted Landfill at a location emitting methane. In total, 26 VOCs were investigated, including chlorinated methanes, ethanes, ethenes, fluorinated hydrocarbons, and aromatic hydrocarbons. The soil showed a high capacity for methane oxidation resulting in very high oxidation rates of between 24 and 112 microg CH4 g(-1) h(-1). All lower chlorinated compounds were shown degradable, and the degradation occurred in parallel with the oxidation of methane. In general, the degradation rates of the chlorinated aliphatics were inversely related to the chlorine to carbon ratios. For example, in batch experiments with chlorinated ethylenes, the highest rates were observed for vinyl chloride (VC) and lowest rates for trichloroethylene (TCE), while tetrachloroethylene (PCE) was not degraded. Maximal oxidation rates for the halogenated aliphatic compounds varied between 0.03 and 1.7 microg g(-1) h(-1). Fully halogenated hydrocarbons (PCE, tetrachloromethane [TeCM], chlorofluorocarbon [CFC]-11, CFC-12, and CFC-113) were not degraded in the presence of methane and oxygen. Aromatic hydrocarbons were rapidly degraded giving high maximal oxidation rates (0.17-1.4 microg g(-1) h(-1)). The capacity for methane oxidation was related to the depth of oxygen penetration. The methane oxidizers were very active in oxidizing methane and the selected trace components down to a depth of 50 cm below the surface. Maximal oxidation activity occurred in a zone between 15 and 20 cm below the surface, as this depth allowed sufficient supply of both methane and oxygen. Mass balance calculations using the maximal oxidation rates obtained demonstrated that landfill soil covers have a significant potential for not only methane oxidation but also cometabolic degradation of selected volatile organics, thereby reducing emissions to the atmosphere.

Bacteria↗

Methane emissions from beef cattle: effects of fumaric acid, essential oil, and canola oil.

The objective of this study was to identify feed additives that reduce enteric methane emissions from cattle. We measured methane emissions, total tract digestibility (using chromic oxide), and ruminal fermentation (4 h after feeding) in growing beef cattle fed a diet supplemented with various additives. The experiment was designed as a replicated 4 x 4 Latin square with 21-d periods and was conducted using 16 Angus heifers (initial BW of 260 +/- 32 kg). Treatments were: control (no additive), fumaric acid (175 g/d) with sodium bicarbonate (75 g/d), essential oil and spice extract (1 g/d), or canola oil (4.6% of DMI). The basal diet consisted of 75% whole-crop barley silage, 19% steam-rolled barley, and 6% supplement (DM basis). Four large chambers (2 animals fed the same diet per chamber) were equipped to measure methane emissions for 3 d each period. Adding canola oil to the diet decreased (P = 0.009) total daily methane emissions by 32% and tended (P = 0.09) to decrease methane emissions as a percentage of gross energy intake by 21%. However, much of the reduction in methane emissions was due to decreased (P < 0.05) feed intake and lower (P < 0.05) total tract digestibility of DM and fiber. Digestibility of all nutrients was also lowered (P < 0.05) by feeding essential oil, but there were no effects on ruminal fermentation or methane emissions. In contrast, adding fumaric acid to the diet increased total VFA concentration (P = 0.03), increased propionate proportions (P = 0.01), and decreased the acetate:propionate ratio (P = 0.002), but there was no measurable effect on methane emissions. The study demonstrates that canola oil can be used to reduce methane losses from cattle, but animal performance may be compromised due to lower feed intake and decreased fiber digestibility. Essential oils had no effect on methane emissions, whereas fumaric acid caused potentially beneficial changes in ruminal fermentation but no measurable reductions in methane emissions.

Animal Feed↗

Methane production and release from two New England peatlands.

The rate of methane production and release to the atmosphere was determined for two New England peat bogs. Methane production rates from peat sediments, which were measured down to depths of 150 cm, ranged from 1 to 15 micromoles per liter per hour. The highest rates of methane production occurred at depths of 60-100 cm. Methane release from these same peats was quantified from various habitats on the bog using gas collection chambers. The chambers enclose a two-liter volume and cover an area of 0.02 m2. Methane accumulation in the chambers was measured for periods of up to 18 days. Methane release was related to pH and habitat zone. The lowest rates of methane release were from those portions of the bogs that had pH values below 5.0. Peak methane release occurred during or immediately after ice melt in both wetlands with release rates as high as 34 mmoles/m2/d. The overall estimate of yearly release of methane from these bog systems is 2,900 and 14,900 moles per year for Arcadia and Hawley Bogs respectively. Both of these bogs have pH environments close to the lowest limit for methanogenesis, and small differences in pH values can have a large impact on both the rate of methane production and the rate of methane release to the atmosphere.

Anaerobiosis↗

[Biogeochemical processes of methane cycle in the soils, swamps and lakes of Western Siberia].

The biogeochemical processes of methane production and oxidation were studied in the upper horizons of tundra and taiga soils and of raised bogs and lake bottom sediments nearby the Tarkosalinsk gas field in western Siberia. Both in dry and water-logged soils, the total methane concentration (in soil particles and gaseous phase) was an order of magnitude higher than in the soil gaseous phase alone (22 and 1.1 nl/cm3, respectively). In bogs and lake bottom sediments, methane concentration was as high as 11 microliters/cm3. Acetate was the major precursor of the newly formed methane. The rate of aceticlastic methanogenesis reached 55 ng C/(cm3 day), whereas that of autotrophic methanogenesis was an order of magnitude lower. The most active methane production and oxidation were observed in bogs and lake sediments where the delta 13C values of CO2 were inversely related to the intensity of bacterial methane oxidation. Methane diffusing from bogs and lake bottom sediments showed delta 13C values ranging from -78 to -47@1000, whereas the delta 13C value of carbon dioxide ranged from -18 to -6@1000. In these ecosystems, methane emission comprised from 3 to 206 mg CH4/(m2 day). Conversely, the dry and water-logged soils of tundra and taiga took up atmospheric methane at a rate varying from 0.3 to 5.3 mg CH4/(m2 day). Methane consumption in soils was of biological rather than of adsorptive nature. This was confirmed by the radioisotopic method and chamber experiments, in which weighting of methane carbon was observed (the delta 13C value changed from -51 to -41@1000).

Bacteria↗